Methods for determining tyrosine-DNA phosphodiesterase activity

Abstract

The present invention provides human tyrosine-DNA phosphodiesterases (TDPs). In particular, the present invention provides novel recombinant nucleic acids and proteins, including mutant TDPs, vectors, and TDP-producing cells, as well as co-factors for enzyme activity. The present invention further provides methods for high through-put enzymatic assay systems utilizing the TDPs of the present invention.

We claim:  
     
       1. A method of determining tyrosine-DNA phosphodiesterase activity, comprising: 
       a. providing:  
       i. tyrosine-DNA phosphodiesterase:  
       ii. p-nitrophenyl thymidine-3′-phosphate; and  
       iii. manganese cation:  
       b. combining said tyrosine-DNA phosphodiesterase, said p-nitrophenyl thymidine-3′-phosphate and said manganese cation under conditions wherein the tyrosine-DNA phosphodiesterase reacts with the p-nitrophenyl thymidine-3′-phosphate to release t,-nitrophenyl; and  
       c. detecting said p-nitrophenyl, wherein said tyrosine-DNA phosphodiesterase is human tyrosine-DNA phosphodiesterase comprising amino acid sequence SEQ ID NQ:2.  
     
     
       2. The A method of determining tyrosine-DNA phosphodiesterase activity, comprising: 
       a. providing:  
       i. tyrosine-DNA phosphodiesterase:  
       ii. p-nitrophenyl thymidine-3′-phosphate; and  
       iii. manganese cation:  
       b. combining said tyrosine-DNA phosphodiesterase, said p-nitrophenyl thymidine-3′-phosphate and said manganese cation under conditions wherein the tyrosine-DNA phosphodiesterase reacts with the p-nitrophenyl thymidine-3′-phosphate to release p-nitrophenyl; and  
       c. detecting said p-nitrophenyl,  
       wherein said tyrosine-DNA phosphodiesterase has an amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:8, 10, 12 and 14. 
     
     
       3. A method of determining tyrosine-DNA phosphodiesterase activity, comprising: 
       a. providing:  
       i. tyrosine-DNA phosphodiesterase; and  
       ii. n-nitrophenyl thymidine-3′-phosphate;  
       b. combining said tyrosine-DNA phosphodiesterase and said p-nitrophenyl thymidine-3′-phosphate under conditions wherein the tyrosine-DNA phosphodiesterase reacts with the n-nitrophenyl thymidine-3′-phosphate to release p-nitrophenyl; and  
       c. detecting said p-nitrophenyl;  
       wherein said tyrosine-DNA phosphodiesterase is human tyrosine-DNA phosphodiesterase comprising amino acid sequence SEQ ID NO:2. 
     
     
       4. A method of claim determining tyrosine-DNA phosphodiesterase activity, comprising: 
       a. providing:  
       i. a tyrosine-DNA phosphodiesterase; and  
       ii. p-nitrophenyl thymidine-3′-phosphate;  
       b. combining said tyrosine-DNA phosphodiesterase and said p-nitrophenyl thymidine-3′-phosphate under conditions wherein the tyrosine-DNA phosphodiesterase reacts with the n-nitrophenyl thymidine-3′-phosphate to release p-nitrophenyl; and  
       c. detecting said p-nitrophenyl,  
       wherein said tyrosine-DNA phosphodiesterase has an amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:8, 10, 12 and 14. 
     
     
       5. A method of determining modulation of tyrosine-DNA phosphodiesterase activity; comprising: 
       a. providing:  
       i. tyrosine-DNA phosphodiesterase;  
       ii. a compound suspected of modulating tyrosine-DNA phosphodiesterase activity; and  
       iii. p-nitrophenyl thymidine-3′-phosphate;  
       b. combining said tyrosine-DNA phosphodiesterase and said p-nitrophenyl thymidine-3′-phosphate in the presence and absence of said compound suspected of modulating tyrosine-DNA phosphodiesterase activity under conditions wherein the tyrosine-DNA phosphodiesterase reacts with p-nitrophenyl thymidine-3′-phosphate to release p-nitrophenyl in the presence and absence of said compound suspected of modulating tyrosine-DNA phosphodiesterase activity; and  
       c. detecting said p-nitrophenyl,  
       wherein said tyrosine-DNA phosphodiesterase is human tyrosine-DNA phosphodiesterase comprising amino acid sequence SEQ ID NO:2. 
     
     
       6. A method of of determining modulation of tyrosine-DNA phosphodiesterase activity, comprising: 
       a. providing;  
       i. tyrosine-DNA phosphodiesterase;  
       ii. a compound suspected of modulating tyrosine-DNA phosphodiesterase activity; and  
       iii. p-nitrophenyl thymidine-3′-phosphate;  
       b. combining said tyrosine-DNA phosphodiesterase and said p-nitrophenyl thymidine-3′-phosphate in the presence and absence of said compound suspected of modulating tyrosine-DNA phosphodiesterase activity under conditions wherein the tyrosine-DNA phosphodiesterase reacts with p-nitrophenyl thymidine-3′-phosphate to release p-nitrophenyl in the presence and absence of said compound suspected of modulating tyrosine-DNA phosphodiesterase activity; and  
       c. detecting said p-nitrophenyl  
       wherein said tyrosine-DNA phosphodiesterase has an amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:8, 10, 12 and 14.